This invention relates generally to methods for the synthesis of phosphonate esters. More particularly, the invention provides methods for producing monoesters and diesters of phosphonic acids from condensations of alcohols with alkylphosphonates, such as methyl .alpha.-aminoalkyl-phosphonates. The methods can be carried out in solution phase, but in an important embodiment, the method is carried out on a solid support, such as beads or a glass slide, and used to create large arrays of diverse compounds, such as peptidylphosphonates and polyphosphonates, which can then be conveniently screened for the presence of compounds with desired pharmacological or other properties. The invention therefore relates to the fields of chemistry, enzymology, pharmacology, and medicinal chemistry.
Aminophosphonic acids and aminophosphonates are derivatives of amino acids in which the amino acid carboxyl group has been replaced with a phosphonic acid or phosphonate moiety. In .alpha.-aminophosphonic acids and phosphonates, the .alpha.-carbon is often a chiral center, bearing a phosphonate moiety, an amine moiety, and one or more amino acid side chains. The structure can be represented as follows: ##STR1## wherein R represents any amino acid side chain and R' and R" represent hydrogen (in the case of a phosphonic acid) or a group such as alkyl or aryl (in the case of a phosphonate ester).
.alpha.-Aminophosphonates are used in the synthesis of peptide analogues (peptidylphosphonates) possessing a phosphonate linkage in the place of at least one amide link in the peptide main chain. The phosphonate linkage can impart various useful properties to peptides. Because the phosphonate linkage in a peptidylphosphonate exists as a charged moiety in the peptide backbone, it increases the water solubility of the peptide. Further, the phosphonate linkage can impart protease resistance and thereby increase the serum half-life of many therapeutic peptide. Still further, substitution of the phosphonate linkage for the amide linkage allows additional functionalities to be introduced in otherwise inaccessible regions of naturally occurring peptides. This is because the phosphonate linkages are tetrahedral, while amide linkages are planar. The tetrahedral geometry allows substituents to be placed above and below the plane of the amide linkage of a peptide. Moreover, the tetrahedral configuration of the phosphonate linkage can be exploited to optimize ligand-receptor binding. See Bartlett and Marlowe (1983) Biochemistry 22:4618-24.
Literature examples of specific uses of peptidylphosphonates are numerous. For example, these compounds are recognized as effective transition-state analogue inhibitors for a variety of enzymes, including a number of other proteases and esterases (see, e.g., Morgan et al. (1991) J. Am. Chem. Soc. 113:297 and Bartlett et al. (1990) J. Org. Chem. 55:6268). Peptidylphosphonate esters have been used as nonhydrolyzable analogues of phosphates to inhibit dinucleoside triphosphate hydrolase (see, e.g., Blackburn et al. (1987) Nucl. Acids Res. 15:6991), phosphatidyltransferase (see, e.g., Vargas (1984) Biochim. Biophys. Acta 796:123), and squalene synthetase (see, e.g., Biller et d. (1988) J. Med. Chem. 31:1869). In fact, the most potent non-covalent enzyme inhibitor known is a phosphonyltripeptide inhibitor of carboxypeptidase A, which binds with 11 femptomolar (fM) K.sub.D. See Kaplan et al. (1991) Biochem. 30:8165-8170. In addition, phosphonate esters have been used as haptens for the production of catalytic antibodies possessing esterase activity ( see, e.g., Jacobs et al. (1987) J. Am. Chem. Soc. 109:2174; Tramontano et al. (1986) Science 234:1566; and Pollack et al. (1986) Science 234:1570; see also, U.S. patent application Ser. No. 858,298, filed Mar. 26, 1992). Some peptidylphosphonates analogues are commercially available therapeutics. For instance, the drugs Monopril and Fosinopril are available from Bristol Myers, Squibb (Evansville, Ind.).
.alpha.-Aminophosphonates are a member of a broader class of compounds, phosphonic acid monoesters. These monoesters have typically been synthesized by one of two methods. In the first, a monomethyl alkylphosphoryl chloride is treated with an alcohol. Selective deesterification of the methyl ester yields the desired product. The monomethyl alkylphosphoryl chloride is produced by the direct action of PCl.sub.5 on a phosphonate diester (see Balthazor et al. (1980) J. Org. Chem. 45:530) or by base hydrolysis of the phosphonate diester followed by reaction with thionyl chloride (see Bartlett et al. (1990) J. Org. Chem. 55:6268).
Alternatively, the direct monoesterification of a phosphonic acid can be accomplished with the appropriate alcohol and condensing reagents (see Gilmore et al. (1974,) J. Pharm. Sci. 63:965), such as 1,3-dicyclohexylcarbodiimide (DCC) or trichloroacetonitrile (see, e.g., Wasielewski et al. (1976) J. Rocz. Chem. 50:1613). A large excess of both the condensing agent and alcohol is required, and yields vary depending on the components being coupled. In addition, forcing conditions are required, such as heating to reflux temperature in a solution of THF with triethylamine and DCC, and to 50.degree. C.-80.degree. C. in pyridine with trichloroacetonitrile, conditions that can lead to decomposition of starting material. In addition, product yields are extremely sensitive to steric encumbrance from the reacting components, and in some cases, no product formation is observed.
The Mitsunobu reaction is a mild and effective method utilizing the redox chemistry of triphenylphosphine and a dialkylazodicarboxylate to condense an acidic reagent with primary and secondary alcohols (see Mitsunobu, (1981) Synthesis 1-28). Mitsunobu does not describe the reaction of phosphate esters with secondary or tertiary alcohols or the reaction of phosphonates with alcohols. The Mitsunobu reaction has been used with carboxylic acids, phenols, and phosphates as the acidic component, but only one example using a phosphonic acid has been described.
In this example (see Norbeck et al. (1987) J. Org. Chem. 52:2174), a monomethyl phosphonate and a primary alcohol were reacted under modified Mitsunobu conditions (triphenylphosphine and diisopropylazodicarboxylate) to produce a phosphonate diester. Demethylation using triethylamine and thiophenol produced the phosphonate monoester. Yields, however, were low for the condensation of a phosphate monoester with even a primary alcohol. Only moderate yields were obtained with elevated temperatures or with hexamethylphosphorous triamide (HMPT) as the solvent.
Recently, innovative combinatorial strategies for synthesizing large numbers of polymeric compounds on solid supports have been developed. One such method, referred to as VLSIPS.TM. ("Very Large Scale Immobilized Polymer Synthesis"), is described in U.S. patent application Ser. No. 07/805,727, filed Dec. 6, 1991, which is a continuation-in-part of Ser. No. 07/624,120, filed Dec. 6, 1990, which is a continuation-in-part of U.S. Pat. No. 5,143,854, which is a continuation-in-part of Ser. No. 07/362,901, filed Jun. 7, 1989, and now abandoned. Such techniques are also described in PCT publication No. 92/10092. Related combinatorial techniques for synthesizing polymers on solid supports are discussed in U.S. patent application Ser. No. 07/946,239 filed Sep. 16, 1992which is a continuation-in-part of Ser. No. 07/762,522 filed Sep. 18, 1991 and U.S. patent application Ser. No. 07/980,523 filed Nov. 20, 1992 which is a continuation-in-part of Ser. No. 07/796,243 filed Nov. 22, 1991. Briefly, a combinatorial synthesis strategy is an ordered strategy for parallel synthesis of diverse polymer sequences by sequential addition of reagents to a solid support.
To synthesize phosphonates, and particularly peptidylphosphonates, on a solid support, one needs a general method for introducing the phosphonic acid monoester functionality. However, the phosphonate synthesis procedures described above generally have not proved easily modified for solid phase applications, which presents a significant problem for the medicinal chemist. Thus, there remains a need for chemical synthesis methods for use in the VLSIPS.TM. method to generate and screen large numbers of peptidylphosphonates and other compounds containing phosphonate ester building blocks. This method should be compatible with a variety of functional groups and consistently produces high yields of the desired compounds and that can be applied to solution as well as solid phase chemistry. The present invention meets these needs.